Supports for the parallel identification and transcription profiling of polynucleic acids

ABSTRACT

The invention relates to a support. Oligonucleotides or polynucleotides are covalently bound with the 5′- or 3′-termination on least one main surface of said support via bifunctional spacers and bifunctional linkers. The support is characterized in that the oligonucleotides or polynucleotides which are covalently bound with the 5′- or 3′-termination via bifunctional spacers and bifunctional linkers comprise 200 to 600 bp, and the oligonucleotides or polynucleotides can be obtained by using a method which comprises the following steps: Selecting homologous regions of mRNA of a target species and of at least one model species; selecting amplification primers which permit the amplification of 200 to 600, preferably 200 to 400 bp long nucleic acids from the homologous regions of both the mRNA of the target species and the mRNA of at least one model species, whereby the amplification primers optionally comprise a maximum of 1 mismatch per 6 nucleic acids of the amplification primer; immobilizing the nucleic acids on at the least one main surface of the support, said nucleic acids being obtained from the corresponding 200 to 600 bp long nucleic acids which are amplified for the target species or for the at least one model species by amplifications using the amplification primers.

The present invention relates to a support comprising oligo- orpolynucleotides covalently linked at their 5′- or 3′-termini to at leastone major surface of said support through bifunctional spacers andbifunctional linkers, the use of the support according to the invention,the preparation of the support according to the invention, and a methodfor establishing transcription profiles.

Analyses performed on a molecular-biological level are increasinglygaining importance. In most cases, a mixture of nucleic acids to beanalyzed is hybridized with so-called probes and characterized in suchmethods. Especially for problems in which a large number of polynucleicacids of different kinds are to be detected simultaneously, there arebottlenecks in the method. It is attempted to perform a large number ofanalytical steps within a short period of time, especially by a paralleloperation of the process. There is often a problem in that supportsystems on which the hybridization experiments can be performed have alimited space capacity. Therefore, it is attempted to solve theseproblems by using supports on which a large number of samples can beplaced. In particular, the prior art describes supports which havemicrometer or nanometer compartments for receiving correspondingly smallvolumes of the analytes which are mostly in solution. Appropriatesupport systems can be obtained, for example, by etching the surfaces ofwafers made of silicon.

E. M. Southern (E. M. Southern et al. (1992), Nucleic Acids Research 20:1679 to 1684, and E. M. Southern et al. (1997), Nucleic Acids Research25: 1155 to 1161) describes the preparation of so-called oligonucleotidearrangements by direct synthesis on a glass surface derivatized with3-glycidoxypropyltrimethoxysilane and then with a glycol.

The publication by S. P. A. Fodor (A. C. Pease et al. (1994), Proc.Natl. Acad. Sci. USA 91: 5022 to 5026) relates to a similar method. Thein situ oligonucleotide synthesis described therein is performed byfully automated light-controlled combinatorial chemistry. The directsynthesis of oligonucleotides on a glass support allows a maximum lengthof about 30 bases. Ensuring a correct course of the synthesis for anindividual sequence of longer oligonucleotides, if at all possible,involves an expenditure which is no longer justifiable. As a guide DNA,these oligonucleotides allow the hybridization of only a rather shortlength of the analyte nucleic acid. To circumvent this drawback, severaloligonucleotides are synthesized as guide DNAs for each nucleic acid tobe analyzed. This results in higher demands for space and thus a largersample volume of the analyte nucleic acid. Further, the small length ofthe oligonucleotides does not preclude cross hybridizations withdifferent analyte nucleic acids. This makes an unambiguous assignment ofthe recorded signals difficult.

For the preparation of so-called DNA chips, P. O. Brown (DeRisi et al.(1997), Science 278: 680-686) discloses polylysine-coated glass surfacesto which minute DNA quantities are applied dropwise by capillarytechniques. However, the immobilization of the guide DNA on a polylysinesurface adversely affects hybridization and thus considerably increasesthe detection limit and reduces the reliability in the detection of theanalyte nucleic acids.

L. M. Smith (Z. Guo et al. (1994), Nucleic Acids Research 22: 5456-5465)describes a technology for the immobilization of oligonucleotides inwhich oligonucleotides are derivatized with a 5′-terminal amino groupand then applied to a glass surface derivatized with3-aminopropyltrimethoxysilane and then with 1,4-phenyldiisothiocyanate.While the chemistry used for the immobilization of the oligonucleotidesavoids the drawbacks of the previously described systems, there arestill the previously mentioned drawbacks caused by the use of shortoligonucleotides as the guide DNA.

Such systems can be prepared only with a high expenditure usually, andoften fail to reach a satisfactory capacity of sample compartments whichwould be necessary for appropriate parallelization.

In addition, the use of complete cDNAs is not advisable. On the onehand, cross-reactions will occur in highly homologous gene families, andon the other hand, the cDNAs contain repetitive elements which mayresult in non-specific hybridizations. This may result in artifacts. Incomparisons between different species, the mentioned artifacts mayresult in a severe limitation of the method.

Thus, it has been the object of the present invention to provide asupport which avoids the mentioned drawbacks of the prior art. Inparticular, the support according to the invention should be able tobind nucleic acids, preferably having a defined sequence and, ifpossible, the same length, in high densities and allow a high level ofparallelization of samples to be examined.

According to the invention, this object is achieved by a support havingthe features of claim 1. Preferred embodiments of the support accordingto the invention are found in the dependent claims. The presentinvention also relates to a method for the preparation of the supportaccording to the invention and its use. The provision of the supportaccording to the invention enables a novel and inventive method whichadvantageously enables the quantification of transcription profiles.

FIG. 1 shows a reaction scheme in which the chemical derivatization ofthe solid phase surface and coupling of the guide DNA are illustrated.

FIG. 2 shows a comparison of the expression levels of 72 different genesin two similar, but differently labeled wild type mouse brain RNAsamples, FIG. 2a relating to a sample labeled with Cy3-dCTP and FIG. 2brelating to one labeled with Cy5-dCTP. FIG. 2c shows signal intensitieswt/wt of the two fluorescence-labeled samples in a double-logarithmicdot diagram. FIG. 2d shows the expression quotient wt/wt, the ratio ofCy3-labeled to Cy5-labeled sample being illustrated as a semilogarithmicbar diagram.

FIG. 3 shows a comparison of the expression levels of 72 different genesin a wild type mouse brain RNA sample and a mutant (PLP^(−/−) MBP^(−/−))mouse brain RNA sample. FIG. 3a relates to the expression profilesobtained with Cy3-dCTP-labeled nucleic acids, and FIG. 3b shows theexpression profiles obtained with Cy5-dCTP-labeled nucleic acids. FIG.3c shows the corresponding signal intensities as in FIG. 2c. FIG. 3dshows normalized expression quotients wt as in FIG. 2d.

The support according to the invention comprising oligo- or polynucleicacids covalently linked to at least one major surface of thesubstantially planar support has a reactive group on the major surfaceof the support, which reactive group has reacted with a bifunctionalspacer to form a covalent bond between a functional group of the spacerand the reactive group of the major surface of the support. “Majorsurface” means any surface which has a sufficient dimension to receive anumber of samples necessary for the use of the support.

The second functional group of the bifunctional spacer has reacted witha functional group of a bifunctional linker, and the second functionalgroup of the bifunctional linker has reacted with the oligo- orpolynucleotide to be covalently linked (guide nucleic acid) to form acovalent bond at the 5′- or 3′-terminus of said oligo- orpolynucleotide.

The support according to the invention is characterized in that saidoligo- or polynucleic acids covalently linked at their 5′- or 3′-terminithrough bifunctional spacers and bifunctional linkers have a length offrom 200 to 600 bp. The oligo- or polynucleic acids can be obtained by amethod comprising the following steps:

selection of homologous regions of mRNA from a target species and atleast one model species;

selection of amplification primers allowing the amplification of nucleicacids having a length of from 200 to 600 bp, preferably from 200 to 400bp, from the homologous regions of both the mRNA from said targetspecies and the mRNA from said at least one model species, theamplification primers optionally having a maximum of 1 mismatch per 6nucleic acids of the amplification primer;

on said at least one major surface of the support, immobilization of thenucleic acids obtained by amplifications of corresponding nucleic acidshaving a length of from 200 to 600 bp for said target species or said atleast one model species using the amplification primers.

Preferably, the polynucleotide is an RNA, DNA or PNA. The supportpreferably consists of a glass or another material mainly consisting ofsilica. Preferably, said bifunctional spacer bonded to the major surfaceof the support according to the invention has the following structure:

(XO)₃—Si—Y—Nu,

wherein

X=C₁-C₃ alkyl,

Y=C₂-C₄ alkylene,

Nu=a nucleophilic group such as —NH₂, —NHR, with

R=—CH₂—CH₂—NH₂, —CH₂—CH₂—NH—CH₂—CH₂—NH₂, —CO—NH₂ or SH.

Particularly preferred is a spacer having the structure

Me₃OS₁—CH₂—CH₂—CH₂—NH₂.

Preferably, said bifunctional linker is selected from the group of rigidhomobifunctional linkers consisting of:

2,7-substituted fluorene, 2,6-substituted naphthalene, 2,6-substitutedanthracene, 2,7-substituted phenanthrene, 4,4′-substituted biphenyl,4,4′-substituted benzoin (C₆H₅—CO—CH(OH)—C₆H₅), 4,4′-substituted benzil(C₆H₅—CO—CO—C₆H₅), 4,4′-substituted benzophenone (C₆H₅—CO—C₆H₅),4,4′-substituted diphenylmethane (C₆H₅—CH₂—C₆H₅), 4,4′-substitutedstilbene (C₆H₅—CH═CH—C₆H₅), 1,3-substituted allene (CH₂═C═CH₂),1,4-disubstituted benzene.

Especially preferred is a linker having the following structure:

S═C═N-phenylene-N═C═S.

The use of rigid bifunctional linkers has the advantage thatsubstantially only one of the two groups reacts with the surface of thesupport.

As functional groups with which said homobifunctional linker issubstituted, there are preferred:

aldehydes and ketones, isocyanates, isothiocyanates, carboxylic acids,carboxylic acid derivatives:

a) carboxylic acid esters: generally the readily available methyl andethyl esters. However, activated esters, such as esters of p-nitrophenolor N-hydroxysuccinimide, should be more suitable;

b) carboxylic acid chlorides (R—COCl);

c) carboxylic acid azides (R—CON₃);

d) mixed anhydrides with carbonic acid monoester (R—CO—O—COR′).

The support according to the invention preferably has an oligo- orpolynucleotide covalently bonded to said bifunctional linker, saidcovalent bonding being effected through a primary amino group attached,synthetically or by a PCR reaction, on the 3′- or 5′-terminus through analkane having a length of from 4 to 30 methylene groups or through apolyether with from 2 to 20 repeating units.

DNA fragments having a length of from 200 to 400 bp are preferred guideDNAs for the following reasons: The length and the thus determinedmelting temperature are sufficient to ensure non-redundant hybridizationwith a high reliability for a careful selection and maximum complexityas exhibited by the human genome.

The shortest cDNAs known are in a range of about 200 bp. Thus, all cDNAsof a cDNA population to be analyzed can be bound to the solid phasecompletely or as fragments of 200 to 400 bp. In the hybridization with alabeled analyte nucleic acid, the similar length of all DNAs appliedresults in hybridization signals which are not adversely affected by thelength or different hybridization kinetics of the guide nucleic acid.

The sequences of the guide DNAs to be applied are preferably searchedindividually, e.g., using a software prepared expressly for thatpurpose, especially in the publicly available gene data bases.Preferably, the guide DNA is to be tested for non-redundancy. Thus, itis essentially excluded that a guide DNA will hybridize with severalanalyte nucleic acids and thus can result in false positive signals. Theguide DNA is amplified from total RNA according to common protocols,e.g., using RT-PCR and sequence-specific primers.

The sequence-specific primers are preferably selected to be suitable forthe amplification of the desired guide nucleic acid from differentspecies, such as human and murine. In this process too, a length of theguide nucleic acid of from 200 to 400 bp has proven useful. The lengthis sufficient to define primers having a length of from 18 to 22 bp inabout 70 to 80% of the instances, which primers allow the amplificationof the guide nucleic acid of both species with a maximum of 3 mismatchbases.

As the support, per se known glass microscope slides are preferablyemployed. In contrast to nylon membranes which are often used, slideshave an advantage, for example, in that they are substantially moreeasily processed and subsequently washed free from non-specifichybridization signals due to their rigidity and the fact that glass isinert towards most reagents. Further, a great advantage resides in thefact that glass can be used for fluorescence-based analysis.

Especially the use of piezoelectric nanodispensers for applying theguide DNA to the solid phase enables very exact dosage, which ispreferred for a reliable quantification of the analyte DNA, and thelatter is directly related with a reproducible amount of guide DNA. Thepossibility of applying drop volumes of 0.1 nl allows to arrange 100,000different guide DNAs on the surface of a slide (76×26 mm), for example.Thus, it becomes possible to detect very small quantities of nucleicacids.

The immobilization of the guide DNA according to the invention throughthe reaction of an isothiocyanate with a primary amine to yieldN-substituted thioureas is advantageous. Both the chemicals and theamino-modified 5′-oligonucleotide primers for the synthesis of DNAs areinexpensive as compared with other synthetic methods which are based onphosphoramidite chemistry. N-substituted thioureas provide stablebonding. The DNAS covalently linked by the method herein described arenot separated from the solid phase even by several hours of boiling inwater. Thus, the DNA chips may also be accessible to repeated use. Withother chips, this is not possible because, inter alia, the washingconditions for removing specific and non-specific analyte DNAs asrequired for regeneration cannot be selected stringent enough due to theinstability of the solid phase bonding.

The specific bonding of the DNA through its 5′-end or 3′-endsubstantially ensures that almost the whole guide DNA is available forhybridizing with the analyte DNA and is not adversely affected bynon-specific binding to the surface. Due to its rather rigid structureand negative charge, the DNA double helix will become alignedperpendicular to the solid phase and thus enable a maximum density ofguide DNA to be applied.

The specific and monovalent binding of the guide DNA to the solid phasenot least allows to control the quantity of DNA applied via thederivatized isothiocyanate groups available.

The present invention also relates to the use according to the inventionof a support according to the invention in a method for identifying andquantifying (assaying) polynucleotides by labeling the polynucleotidesto be analyzed and subsequently hybridizing them on the support.

The method according to the invention for establishing transcriptionprofiles comprises the following steps:

homologous regions of mRNA from a target species and at least one modelspecies are selected;

amplification primers allowing the amplification of nucleic acids havinga length of from 200 to 600 bp, preferably from 200 to 400 bp, from thehomologous regions of both the mRNA from said target species and themRNA from said at least one model species are selected, theamplification primers having a maximum of 1 mismatch per 6 nucleic acidsof the amplification primer;

corresponding nucleic acids having a length of from 200 to 600 bp forsaid target species or said at least one model species are amplified byamplifications using the amplification primers, and the nucleic acidsobtained are immobilized on said at least one major surface of thesupport;

said at least one support is incubated with a DNA or RNA sample to beanalyzed, and the quantity of bound DNA or RNA is determined.

To enable the establishing of a transcription profile, i.e., aqualitative and quantitative analysis of gene expression, the nucleicacid to be analyzed, e.g., RNA or DNA, is labeled. Then, due to thehybridization process, the cDNAs immobilized on the surface are joinedwith the corresponding labeled DNAs or RNAs of the sample to beanalyzed. This results in the cDNAs on the surface of the support beinglabeled with the corresponding counterpart which has been present in thesample to be analyzed.

Said labeling of the sample to be analyzed can be effected by variousmethods.

For example:

1. agents directly reacting with the RNA or DNA may be used;

2. modified nucleotides can be added enzymatically;

3. the RNA can be transcribed into labeled cDNA by a RT reaction withincorporation of modified nucleotides.

The agents or modified nucleotides may contain elements which are, forexample, radioactive or can be excited for fluorescence or luminescence,so that direct measurement is possible with a suitable detector device.However, they may also serve as linkers or haptenes to enable asubsequent coupling with a second labeled molecule.

By the use of different fluorescence signals, different samples to beanalyzed may also be simultaneously applied to one support forhybridization so that a direct comparison of these different samples onone support is possible. Thus, for example, two different samples can betreated as follows. The nucleic acids present in one sample are labeledwith a first fluorescent compound, and the nucleic acids present in thesecond separate sample are labeled with a second fluorescent compoundwhose emitted fluorescence is distinct from that of the firstfluorescent compound.

In addition to the labeled samples, the hybridization solution containssalts, detergents and unlabeled DNA, so that optimum hybridizationconditions matched to the respective support can be achieved. Prior tothe actual hybridization, it may be appropriate to perform a so-calledprehybridization.

Thus, the corresponding support is incubated with the hybridizationsolution, but without a labeled sample; and the reaction conditions canbe optimized.

After the hybridization reaction, non-specifically bound samplecomponents are separated off by one or, preferably, more washingprocedures. The washing solutions employed contain, in particular,detergents, such as sodium dodecylsulfate, and low amounts of salts. Thewashing procedure(s) are usually performed in a temperature range offrom 20 to 60° C. and over a period of from 5 to 20 minutes.

To establish a transcription profile, the signals recorded by means of asuitable detector device are quantified. For example, the signalintensity directly corresponds to the number of labeled moleculespresent in the hybridization solution and thus to the expression levelof the respective gene in the sample examined. By normalizing thesevalues to an internal or external standard, transcription profiles ofdifferent samples can be compared with each other.

The detection of the hybridization of guide and analyte nucleic acidscan be effected by various methods. One suitable method involves thelabeling of the analyte nucleic acid with fluorescent nucleotides,followed by detection of the hybridization by fluorescence microscopy.For the differential or relative quantification of analyte nucleicacids, a known quantity of reference nucleic acid carrying a secondfluorescence marker is added to the fluorescence-labeled analyte nucleicacid and applied to the immobilized guide DNA for hybridization. Arelative quantification of the analyte nucleic acid is effected bycomparing the detected signal intensities.

The method according to the invention for the preparation of a supportaccording to the invention comprises the following steps.

The bifunctional spacer in a polar aprotic solvent is applied to themajor surface of the support, and any excess (unreacted) spacer issubsequently removed. The bifunctional spacer is applied to the majorsurface of the support, for example, in a 95% by volume acetone/watermixture. After the optional washing steps, the support is preferablydried, especially by heating.

The bifunctional linker is dissolved in an essentially anhydrous polaraprotic solvent and reacted with the spacer bound to the major surface.The linker should preferably be present in a low concentration, forexample, at around 0.5% by weight, in the polar aprotic solvent. In thiscase, a solvent system with 10% pyridine/dimethylformamide (% by volume)may be used, for example. The reaction time depends on the reactivity ofthe bifunctional spacer or bifunctional linker and may be as long asseveral hours at room temperature or increased temperature. In thisstate, the support can be cooled and stored in a dry place for severalmonths.

In a further reaction step, the oligo- or polynucleotide modified withan amino group at its 5′- or 3′-terminus through an alkylene group istaken up in a buffer. In particular, a basic buffer, for example, acarbonate buffer, may be conveniently employed. The mixture is incubatedon the previously prepared support for binding the oligo- orpolynucleotide to a free group of the bifunctional linker. This may bedone, in particular, for a period of several hours in a vapor-saturatedatmosphere. Thereafter, any unreacted groups of the bifunctional linkerare removed. In particular, amines, such as ethanolamine orhydroxylamine, are used for this purpose. These are typical blockingreactions for reactive groups per se known to those skilled in the art.

Thereafter, the oligo- or polynucleotide bound to the support isdenatured. For denaturing, for example, the support is boiled inbidistilled water together with the covalently bound polynucleotide. Tomaintain the denatured state, the support may be washed with purealcohol and then stored in a cool and dry place.

The invention is described in more detail by the following furtherexplanations.

FIG. 1: Chemical derivatization of the solid phase surface and covalentcoupling of the guide DNA.

Cleaning of the glass slides: 76×26 mm, clear white glass, no coating,frosted edge or marking area; e.g., Fisher Scientific under thedesignation “Glass microscope slides, clear white, with cut edges”:agitating the slides for two hours in a solution of 2 N NaOH in 70%EtOH, three washes with completely desalted (and bidistilled) water andone wash with acetone.

Coating: The slides are immersed in a 1% solution of3-aminopropyltrimethoxysilane (APTMS) in 95% acetone/water for twominutes, followed by ten washes in acetone for five minutes each anddrying at 110° C. for 45 minutes.

Derivatization of the coated slides with a linker: The slides areimmersed in a solution of 0.2% by weight 1,4-phenyldiisothiocyanate(PDC)/10% by volume pyridine/dimethylformamide for two hours andsubsequently washed with methanol and acetone.

Application of the guide DNA for covalent coupilng: 0.1 nl volumes ofPCR fragments are applied to defined positions on the slides using ananodispenser, the slides are incubated at 37° C. in a moist atmospherefor at least one hour, followed by one rinse with 1% NH₄OH and threerinses with water, cooling and storing in a dry place.

For separating the DNA complementary strand from the template strand,the slides are incubated at 96° C. in completely desalted water for tenminutes, rinsed with 96% ethanol and subsequently dried at roomtemperature. The slides are now ready for hybridization with the analytenucleic acid.

According to the invention, the following procedure may also be used.

A glass surface which has not been processed for use as a microscopeslide may be employed.

The density of the derivatizable amino groups on the glass surface canbe varied between 0.1 and 10% through the concentration of the APTMSsolution, whereby different densities of coupled guide DNA can beadjusted.

The density of the derivatizable amino groups can further be controlledby a mixture of APTMS/propyltrimethoxysilane (PTMS) orAPTMS/tetramethoxysilane (TeMS) in a ratio of from 1:10 to 10:1.

Prior to the derivatization of the amino groups with PDC, the boundAPTMS molecules can be cross-linked: 30 minutes at 90° C. with 5% APTMSor PTMS or TeMS in water; pH 5.5 to 5.8.

As described above for APTMS, the concentration of PDC can be variedbetween 0.04% and 1% to thereby adjust the density of the linkers forreceiving the guide DNA according to need.

Instead of PDC, other molecules substituted with diisocyanates can alsobe used. PDC and other rigid homobifunctional linkers have the advantagethat the cross-linking of neighboring amino groups is stericallyhindered. In order to obtain a larger distance between the supportsurface and the DNA, it may be advantageous to insert longer linkermolecules between the derivatized surface and the DNA or to use a chainof several units of short linker molecules.

The generation of the PCR fragments is preferably effected bytranscription of the information of an mRNA into DNA using reversetranscriptases. This DNA is then amplified by polymerase chain reaction(PCR). For both enzymatic processes, oligonucleotide primers which willhybridize to a template and serve as synthesis initiators for therespective polymerase are required, inter alia.

A list of genes is established whose parallel identification andquantification is of interest. This list is used as an input for aprogram created for this purpose. By means of the program, the sequencesof the genes to be analyzed are taken from, for example, a publiclyaccessible gene data base, and oligonucleotide pairs are designed whichgive each a specific amplification of 200 to 400 bp fragments from eachgene. The oligonucleotides are synthesized, and the RT PCR is performedaccording to a protocol per se known to those skilled in the art. Forseparating non-incorporated nucleotides and oligonucleotides, the PCRfragments are precipitated with ethanol, and a concentration of 10 to1000 ng/μl, preferably 50 to 500 ng/μl, is adjusted with 100 mM sodiumcarbonate/sodium hydrogencarbonate solution, pH 9.

In the following two Examples relating to the establishing of atranscription profile, supports were prepared by the method according tothe invention. Seventy-two different murine cDNAs were generated usingthe corresponding specific primers (oligonucleotides) in an RT PCR frommurine brain total RNA, applied to the derivatized glass surface andcovalently bound. Each cDNA was applied twice each in four quadrants,i.e., a total of eight times. Two nanoliters each of the correspondingcDNA having a concentration of 100 ng/μl was applied using a dispensingautomatic. The diameter of the dried-on samples was 350 μm each, and thedistance from one center to the next for two neighboring samples was0.750 μm.

In the Examples, an expression profile of wild type and mutant mousebrain samples is established. Thus, the brain of 18-day-old wild typeand mutant mice (MBP^(−/−)/PLP^(−/−)) was dissected, and the total RNAextracted. For labeling the respective samples, the mRNA was isolatedfrom 100 μg each of total RNA and transcribed into the correspondingcDNA using a reverse transcriptase. During this process,fluorescence-labeled nucleotides (Cy3-dCTP or Cy5-dCTP) wereincorporated. The labeled samples were purified, adjusted to the optimumconditions for hybridization and concentrated to a volume of 20 μl.

Prior to the actual hybridization reaction, the support was subjected toprehybridization. Thus, 20 μl of a salt/detergents/unlabeled DNAsolution was applied to the support and provided with a cover slip.After two hours of incubation at 62° C. in a moist chamber sealedtowards the exterior, the reaction solution was cooled to 20° C., thecover slip was removed, and a 20 μl drop of the actual hybridizationsolution was added. Again, the solution was provided with a cover slipand incubated at 62° C. in the mentioned chamber for 12 hours.Subsequently, non-specifically bound samples were washed off the supportwith two different washing solutions. The detection of the signals waseffected using the laser scanning device “ScanArray 3000” supplied byGeneral Scanning, Watertown, Mass., USA. Commercially available software(“ImaGene” of BioDiscovery, Inc., Los Angeles, Calif., USA) was used forevaluating the signals.

EXAMPLE 1

mRNA was isolated twice from the same total RNA and labeled withCy-3-dCTP or Cy5-dCTP. Both samples were applied to a support forhybridization as described above. FIG. 2 shows the images recorded forthe Cy3-labeled sample (a) and for the Cy5-labeled sample (b).

The signal intensity values from the two fluorescence-labeled samples asobtained using the ImaGene software are represented in FIG. 2c in adouble-logarithmic dot diagram. Each dot in the diagram represents onecDNA applied to the support. Since each of the 72 cDNAs was appliedeight times, eight dots are obtained for each cDNA. On the x and y axes,the signal intensities for the Cy3-labeled and Cy5-labeled samples,respectively, can be read for the respective cDNA. The solid lineencloses all dots whose Cy3 and Cy5 signal intensities differ by no morethan a factor of 2. The dotted line forms the boundary for a three-timesdifferential signal intensity. Since the two labeled samples are derivedfrom the same total RNA, all dots are enclosed by the solid line. Theabsolute intensity of the individual dots and thus ultimately theexpression level of the corresponding genes extends through three powersof ten. FIG. 2d shows the ratio of Cy3 to Cy5 of the labeled sample as asemilogarithmic bar diagram. The data are respectively based on the meanvalues from the eightfold applied cDNAs.

EXAMPLE 2

Comparison of the Expression Pattern of a Wild Type Sample with that ofa Mutant Sample

The wild type sample was labeled with Cy3, and the mutant sample waslabeled with Cy5. FIGS. 3a and b again show the recorded images. FromFIG. 3c, it can be seen that many dots are outside the area bounded bythe dotted lines. Thus, the corresponding genes are expressed on levelswhich differ by more than a factor of three. In FIG. 3d, the signalquotients of the mean values are plotted for each cDNA. Genes 1, 6, 33and 39 show the highest differences in expression level. Thus, forexample, gene No. 6 is expressed at an about 100 times higher level inthe wild type sample while gene No. 33, for example, is expressed at anabout 100 times higher level in the mutant sample. The sum of the cDNAsdetectable in this analysis and the related expression levels can bedefined as a transcription profile for the respective sample. Thedifferential expression of some genes as demonstrated in this analysiscan be considered a first indication of a causal relation between thesegenes and the genes mutated in the mutant sample.

What is claimed is:
 1. A support comprising polynucleotides covalentlylinked at their 5′- or 3′-termini to at least one major surface of saidsupport through at least one bifunctional spacer and at least onebifunctional linker, wherein: said polynucleotides have a length of from200 to 600 bp; said bifunctional linker is selected from the group ofrigid homobifunctional linkers consisting of 1,4-disubstituted benzene,2,7-substituted fluorene, 2,6-substituted naphthalene, 2,6-substitutedanthracene, 2,7-substituted phenanthrene, 4,4′-substituted biphenyl,4,4′-substituted benzoin (C₆H₅—CO—CH—(OH)—C₆H₅), 4,4′-substituted benzil(C₆H₅—CO—CO—C₆H₅), 4,4′-substituted benzophenone (C₆H₅),4,4′-substituted diphenylmethane (C₆H₅—CH₂—C₆H₅), 4,4′-substitutedstilbene (C₆H₅—CH═CH—C₆H₅), and 1,3-substituted allene (CH₂═C═CH₂); saidpolynucleotides are covalently bound to a functional group of saidbifunctional linker through a primary amino group attached, on the 3-′or 5′-terminus through an alkane having a length of from 6 to 18methylene groups or though a polyether having from 2 to 20 repeatingunits; and the polynucleotides are prepared by amplification.
 2. Thesupport according to claim 1, wherein said polynucleotide is RNA, DNA orPNA.
 3. The support according to claim 1, wherein said support is madeof glass or another material consisting essentially of silica.
 4. Thesupport according to claim 1, said bifunctional spacer having thestructure (XO)₃Si—Y—Nu, wherein X=C₁-C₃ alkyl, Y=C₂-C₄ alkylene, andNu=a nucleophilic group.
 5. The support according to claim 4, whereinthe nucleophilic group is —NH₂ or —NHR, with R═—CH₂—CH₂—NH₂,—CH₂—CH₂—NH—CH₂—CH₂—NH₂, —CO—NH₂, or SH.
 6. The support according toclaim 1, wherein said spacer is (MeO)₃Si—CH₂—CH₂—CH₂—NH₂.
 7. The supportaccording to claim 1, wherein said rigid homobifunctional linkercomprises functional groups selected from the group consisting of:aldehydes and ketones; isocyanates and isothiocyanates; carboxylicacids; and carboxylic acid derivatives.
 8. The support of claim 7,wherein the carboxylic acid derivatives are selected from the groupconsisting of: a) carboxylic acid esters; b) carboxylic acid chlorides(R—COCl); c) carboxylic acid azides (R—CON₃); and d) mixed anhydrideswith carbonic acid monoester (R—CO—O—COR′).
 9. The support of claim 8,wherein the carboxylic acid esters are selected from the groupconsisting of methyl esters, ethyl esters, activated esters, and estersof p-nitrophenol and p-hydroxysuccinimide.
 10. The support of claim 1wherein the support does not comprise a polyT-spacer.
 11. The support ofclaim 1 wherein the number of different polynucleotides is at least 72.12. The support of claim 11, wherein the number of differentpolynucleotides is at least
 439. 13. A method for identifying andquantifying polynucleotides comprising the steps of: a) labeling thepolynucleotides to be analyzed; b) hybridizing the polynucleotides onthe support according to claim 1; and c) detecting hybridized labelednucleic acids; wherein steps (a) and (b) are performed in any order. 14.A method for establishing transcription profiles comprising: a)selecting homologous regions of mRNA from a target species and at leastone model species; b) selecting amplification primers from thehomologous regions of both the mRNA from said target species and themRNA from said at least one model species, wherein the amplificationprimers allow amplification of nucleic acids having a length of from 200to 600 bp, and wherein each amplification primer has a maximum of 1mismatch per 6 nucleic acids of the amplification primer; c) amplifying,using the amplification primers, corresponding nucleic acids having alength of from 200 to 600 bp for said target species or said at leastone model species; d) immobilizing the nucleic acids obtained on atleast one support according to claim 1; e) incubating said at least onesupport with a DNA or RNA sample to be analyzed; and f) determining thequantity of bound DNA or RNA.
 15. The method of claim 14, herein thenucleic acids have a length of 200 to 400 bp.
 16. A method forgenerating of a support according to claim 1, comprising: a) applyingthe spacer in a polar aprotic solvent to the major surface of thesupport; b) removing excess unreacted spacer; c) dissolving the linkerin an anhydrous polar aprotic solvent wherein the linker and the spacer,bound to said major surface, react; d) dissolving in a buffer thepolynucleotides modified with an amino group at their 5′- or 3′-terminithrough an alkylene group; e) incubating the polynucleotides on saidsupport to react and bind the polynucleotides to free groups ofbifunctional linkers; f) optionally removing excess free groups of thebifunctional linkers; and g) denaturing the polynucleotides bound to thesupport.